Project summary: We aim to identify the molecular network required for development and survival of mammalian rod photoreceptors. We identified Protein Inhibitor of Activated STAT3 (PIAS3), a transcription co-regulator and site-specific SUMOylase as being strongly expressed in developing rod photoreceptors. We observed that Pias3 overexpression gives rise to an increase in rod photoreceptors, while PIAS3 knockdown produces excess Muller glia. We also identified two transcription factors, Crx and Nr2e3, as Pias3 interacting proteins. We thus hypothesize that PIAS3 plays a critical role in rod development and survival by interacting with photoreceptor-specific transcription factors. We propose to test this hypothesis with a series of experiments. First, we will use fluorescent in situ hybridization and immunocytochemistry to determine if Pias3 is expressed in developing rod photoreceptors and absent from developing cones and mitotic progenitors. Second, based on our preliminary data, we hypothesize that PIAS3 regulates both the fates of differentiating photoreceptors, the kinetics of rhodopsin expression, and the maintenance of newly differentiated photoreceptors. To address these questions, we will perform a detailed analysis of the effects of gain/loss of function of Pias3 using a panel of molecular markers. We will use BrdU-based birthdating to determine whether gain/loss of function of PIAS3 influences the kinetics of rhodopsin expression, and use rhodopsin-based expression constructs to determine whether gain/loss of Pias3 function has distinct effects in developing retina and differentiated photoreceptors. Finally, we will explore the mechanism by which Pias3 acts in retinal development. We hypothesize that the effects of PIAS3 are in part mediated by Crx, Nr2e3 and possibly Stat3. We also hypothesize that Pias3 directly regulates transcription of many rod, cone and possibly Muller glia-specific genes. We test whether PIAS3 interacts with Crx, Nr2e3 or Stat3 in vivo, and whether these factors mediate the effects of PIAS3 in developing retina. Following on from this, we will determine which domains of PIAS3 are required for its activity in the retina, and whether these are also required for interaction with Crx and Nr2e3. Finally, we will determine whether PIAS3 directly regulates expression of rod, cone and Muller-specific genes, and if this requires Crx and Nr2e3. Relevance: The molecular basis of cell specification in the central nervous system is poorly understood, and these studies will provide mechanistic insight into this process. Moreover, mutations in rod-enriched transcription factors very often lead to photoreceptor degeneration and blindness, and we anticipate that this may also hold for Pias3.